Trophic hormones acutely stimulate steroid production by acting on the rate-determining step of steroidogenesis, the transport of the substrate, cholesterol, from intracellular stores to the inner mitochondrial membrane. There, cholesterol will be metabolized to pregnenolone. Using various approaches we demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane and the subsequent steroid biosynthesis. We further demonstrated that PBR functions as a channel specific for cholesterol. It is our hypothesis that the acute (sec to min) hormonal regulation of the PBR receptor complex, preferentially located on the mitochondrial membrane contact sites, is responsible for the hormonal induction of steroidogenesis and that the interaction of the PBR complex with the hormone-induced steroidogenic acute regulatory protein (StAR) is responsible for sustaining steroid production for longer periods of time (hours). In the first aim we will establish the temporal and spatial relationship of PBR and StAR proteins in response to hCG. In the second aim we will examine the structure of the PBR complex in the mitochondrial contact sites isolated from control and LH-treated Leydig cells. In search for the "molecular switch" responsible for the effects of LH/hCG on PBR we identified two candidate PBR-associated proteins (PAPs). In the third aim, we will investigate the role of the PAPs in the hormone-induced changes in PBR, cholesterol transport, and steroidogenesis. Considering the finding that StAR exerts its effect outside the mitochondrion, it is possible that it may act directly on PBR or indirectly via a PAP. In the fourth aim we will examine the StAR-PBR interaction and the functional consequences of this interaction. Our goal is to understand the sequence of events responsible for the induction and maintenance of steroidogenesis by hormones. Our Leydig cell model systems are the MA-10 hormone- responsive cell line, purified rat Leydig cells, the R2C cell line expressing constitutive steroidogenesis, and R2C PBR negative (-) cells, generated by gene targeting.